Inner Nuclear Membrane Asi Ubiquitin Ligase Catalytic Subunits Asi1p and Asi3p, but not Asi2p, confer resistance to aminoglycoside hygromycin B in Saccharomyces cerevisiae

The heterotrimeric Asi ubiquitin ligase (encoded by ASI1, ASI2, and ASI3) mediates protein degradation in the inner nuclear membrane in Saccharomyces cerevisiae. Asi1p and Asi3p possess catalytic domains, while Asi2p functions as an adaptor for a subset of Asi substrates. We hypothesized the Asi complex is an important mediator of protein quality control, and we predicted that Asi would be required for optimal growth in conditions associated with elevated abundance of aberrant proteins. Loss of Asi1p or Asi3p, but not Asi2p, sensitized yeast to hygromycin B, which promotes translational infidelity by distorting the ribosome A site. Surprisingly, loss of quality control ubiquitin ligase Hul5p did not sensitize yeast to hygromycin B. Our results are consistent with a prominent role for an Asi subcomplex that includes Asi1p and Asi3p (but not Asi2p) in protein quality control.

. ASI1 and ASI3 confer resistance to hygromycin B: (A-C) Sixfold serial dilutions of yeast of the indicated genotypes were spotted onto agar plates containing rich growth medium (No Drug) or indicated concentrations of hygromycin B. Plates were incubated at 30°C and imaged after 1-3 days. Experiments were performed in triplicate. (C) "asi2Δ (YKO)" is VJY852 and was obtained from the Yeast Knockout Collection (Tong et al., 2001). "asi2Δ (new clone 1)" and "asi2Δ (new clone 2)" are VJY969 and VJY970, respectively, and were generated for this study.
The aminoglycoside hygromycin B produced by the bacterium Streptomyces hygroscopicus reduces translational fidelity by distorting the ribosome A site, resulting in inaccurately synthesized protein molecules (Brodersen et al., 2000;Ganoza and Kiel, 2001). We previously demonstrated that loss of ER and nuclear PQC ubiquitin ligases Hrd1p, Doa10p, and Ubr1p sensitizes cells to hygromycin B Niekamp et al., 2019;Runnebohm et al., 2020). The extent of Asi's contribution to PQC relative to these enzymes remains unknown.
We hypothesized that Asi is an important mediator of PQC. We predicted that the Asi complex would be required for resistance to conditions expected to increase the abundance of aberrant proteins. To test this, we cultured wild type yeast, yeast lacking genes encoding each subunit of the Asi complex, and a panel of PQC mutant yeast strains in the absence and presence of increasing concentrations of hygromycin B ( Figure 1A). Consistent with previous results, loss of HRD1 or DOA10 sensitized cells to 75 μg/ml hygromycin B, and yeast deleted for UBR1 exhibited sensitivity at concentrations as low as 25 μg/ml. By contrast, deletion in two different genetic backgrounds of the gene encoding PQC ubiquitin ligase Hul5p (Fang et al., 2011;Runnebohm et al., 2020;Sitron and Brandman, 2019) did not sensitize cells to hygromycin B at the concentrations evaluated ( Figure 1A, 1B).
Loss of ASI1 and ASI3 sensitized cells to 75 μg/ml hygromycin B to a similar extent as loss of DOA10 or HRD1 ( Figure  1A). Intriguingly, loss of ASI2 in multiple independently generated yeast strains did not confer a similar growth disadvantage under these conditions ( Figure 1A, 1C). Deletions of ASI genes and HUL5 were validated by PCR. Taken together, our results indicate Asi1p and Asi3p, but not Asi2p, are required for optimal growth in the presence of a compound expected to generate increased numbers of PQC substrates.
The finding that loss of Hul5p does not enhance sensitivity to hygromycin B was surprising, given multiple characterized functions of Hul5p in PQC. Among other roles, Hul5p promotes degradation of substrates that have escaped detection by the ribosome quality control ubiquitin ligase Ltn1p (Sitron and Brandman, 2019) and promotes turnover of misfolded proteins following heat shock (Fang et al., 2011). Loss of Ltn1p sensitizes cells to hygromycin B (Bengtson and Joazeiro, 2010;Crowder et al., 2015). We speculate that a requirement for Hul5p in hygromycin B resistance may become apparent during conditions characterized by elevated cellular dependence on Hul5p, such as compromised Ltn1p function or heat shock.
Multiple lines of evidence suggest that a subcomplex of the Asi ubiquitin ligase including Asi1p and Asi3p (but not Asi2p) mediates PQC degradation of misfolded proteins, potentially in complex with unidentified substrate adaptors. First, as demonstrated here, deletion of ASI1 or ASI3, but not of ASI2, sensitizes cells to conditions expected to increase the abundance of aberrant, mistranslated proteins to an extent similar to that observed following loss of other characterized PQC genes (we note it remains possible that ASI2 is required for optimal growth under different forms of proteotoxic stress, such as elevated temperature). Second, while Asi1p, Asi2p, and Asi3p collaborate to mediate degradation of a host of mislocalized proteins, only Asi1p and Asi3p promote degradation of mutated translocon component sec61-2p (Foresti et al., 2014). Finally, simultaneous deletion of genes encoding Hrd1p, Ire1p (a component of the yeast unfolded protein response), and either Asi1p or Asi3p causes markedly slower growth than concurrent knockout of HRD1, IRE1, and ASI2 (Foresti et al., 2014).
Asi2p function is also dispensable for degradation of some Asi1/3p substrates that do not possess features rendering them predicted PQC substrates (Khmelinskii et al., 2014). Such substrates may expose degradation signals (e.g. when other complex subunits are present in substoichiometric abundance) resembling those of quality control substrates, co-opting a PQC enzyme for regulatory purposes. The precise nature of degradation signal(s) recognized by Asi remains to be resolved.
Yeast growth assay. Yeast growth analysis was performed as described (Watts et al., 2015). Four μl of sixfold serial dilutions were pipetted onto yeast extract-peptone-dextrose medium (Guthrie and Fink, 2004) in the absence or presence of increasing concentrations of hygromycin B (Gibco). Plates were incubated at 30°C and imaged at the indicated times.